Effect of the low molecular weight thymus factor on T-cells in human blood]

Lowmolecular factor of polypeptide nature (thymarin) was extracted from calf thymus. This factor differed by physical and chemical characteristics from thymosin. The percentage content of "active" T-lymphocytes increased under the effect of thymarin in the cultures of lymphoid cells obtained from healthy individuals. The total T-cell count remained unchanged. Thymarin favoured increase of the total T-lymphocyte and "active" T-lymphocyte count in the cultures of cells from patients with chronic inflammatory processes. Cellular immunity reactions were restored and the course of the main disease improved under the effect of parenteral administration of thymarin. It is supposed that T-immunity system incompetence was associated with the inadequate production of the thymus factor capable of restoring the activity of the thymus-dependent lymphocyte population.     

The role of co-inhibitory signals driven by CTLA-4 in immune system

Key players on the immunosurveillance program, T cells are regulated by their surface receptors such as T-cell receptor (TCR), and costimulatory molecules, optimizing T-cell activation. Some of these costimulatory molecules, such as the cytotoxic T-lymphocyte antigen 4 (CTLA-4), induce inhibitory effects on T cells. By "inhibiting the inhibitor" CTLA-4-blocking monoclonal antibodies represent a novel class of weapons against cancers. To better understand the promising future and the limits of this immunotherapy, this review dissects the molecular inhibitory mechanisms induced by CTLA-4 in T cells.     

The complete primary structure of the T -Cell receptor genes from an alloreactive cytotoxic human T-lymphocyte clone

The complete primary structure of the cDNAs encoding the and chains of the T-lymphocyte receptor for antigen from a human alloreactive, cytotoxic T-cell clone, L17, is presented. Sequence analysis of these genes reveals that both are related to immunoglobulins and are composed of variable, diversity (at least in the case of the Ti clone), joining, and constant region sequences. Comparison of the sequence of the -chain cDNA to that of previously sequenced mouse and human cDNAs suggests the presence of human T-cell receptor D-region sequences. Southern blot analysis confirms the finding that these cDNAs represent the functional receptor genes expressed by the L17 cytotoxic T-cell clone. The availability of these full-length T-cell receptor cDNA clones from a human T-lymphocyte clone of known antigen specificity should allow an analysis of the relationship between T-cell receptor structure and function.     

Human Memory Cytotoxic T-Lymphocyte (CTL) Responses to Hantaan Virus Infection: Identification of Virus-Specific and Cross-Reactive CD8+ CTL Epitopes on Nucleocapsid Protein

Hantaan virus, the prototypic member of the Hantavirus genus, causes hemorrhagic fever with renal syndrome in humans. We examined the human memory T-lymphocyte responses of three donors who had previous laboratory-acquired infections with Hantaan virus. We demonstrated virus-specific responses in bulk cultures of peripheral blood mononuclear cells (PBMC) from all donors. Bulk T-cell responses were directed against either Hantaan virus nucleocapsid (N) or G1 protein, and these responses varied between donors. We established both CD4(+) and CD8(+) N-specific cell lines from two donors and CD4(+) G1-specific cell lines from a third donor. All CD8(+) cytotoxic T-lymphocyte (CTL) lines recognized one of two epitopes on the nucleocapsid protein: one epitope spanning amino acids 12 to 20 and the other spanning amino acids 421 to 429. The CTL lines specific for amino acids 12 to 20 were restricted by HLA B51, and those specific for amino acids 421 to 429 were restricted by HLA A1. The N-specific CTL lines isolated from these two donors included both Hantaan virus-specific CTLs and hantavirus cross-reactive CTLs. Responses to both epitopes are detectable in short-term bulk cultures of PBMC from one donor, and precursor frequency analysis confirms that CTLs specific for these epitopes are present at relatively high precursor frequencies in the peripheral T-cell pool. These data suggest that infection with Hantaan virus results in the generation of CTL to limited epitopes on the nucleocapsid protein and that infection also results in the generation of cross-reactive T-cell responses to distantly related hantaviruses which cause the distinct hantavirus pulmonary syndrome. This is the first demonstration of human T-lymphocyte responses to Hantaan virus.     

CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery

BACKGROUND: CD69 is a surrogate marker of T-cell responsiveness to mitogen and Ag stimulus and can be used as a measure of T-lymphocyte activation. Quantitative flow cytometric determination of CD69 expression on T lymphocytes has several advantages over traditional lymphocyte proliferation assays, but this method has not yet been standardized for clinical applications. METHODS: We qualified a commercially available assay using the manufacturer's procedures for measurement of T-cell response to a mitogen (PHA), superantigen (Staphylococcus endotoxin B; SEB) and Ca(2+) ionophore (phorbyl myristate acetate; PMA) with peripheral blood from healthy volunteers. Following this, we tested the usefulness of the assay in determining T-cell responses to PHA and SEB for six immunocompromised patients. RESULTS: Healthy volunteers showed 17-fold increases in T-cell CD69 Ab bound per cell (ABC) with PHA stimulation compared with the baseline. SEB was also an effective T-cell activating agent, increasing CD69 ABC by 5-fold, comparable with results obtained with PMA stimulation. PHA- and SEB-stimulated T-cell CD69 ABC for patients 100 days post-BM transplant were generally below 1 SD of that from healthy volunteers. SEB-stimulated T-cell CD69 expression was significantly depressed for CD8(+) T cells while CD4(+) T-cell responses to SEB were generally within 1 SD of the mean for healthy volunteers. DISCUSSION: These results suggest that quantitative measurement of CD69 surface expression by flow cytometry is a useful diagnostic tool for detailed assessment of T-lymphocyte and subset activation.     

Bioinformatics tools for identifying class I-restricted epitopes

The lack of simple methods to identify relevant T-cell epitopes, the high mutation rate of many pathogens, and restriction of T-cell response to epitopes due to human lymphocyte antigen (HLA) polymorphism have significantly hindered the development of cytotoxic T-lymphocyte (CTL) epitope-based or "epitope-driven" vaccines. Previously, CTL epitopes were mapped using large arrays of overlapping synthetic peptides. The large number of protein sequences available for mapping is now making this method prohibitively expensive and time-consuming. Bioinformatics tools such as EpiMatrix and Conservatrix, which search for unique or multi-HLA-restricted (promiscuous) T-cell epitopes and identify epitopes that are conserved across variant strains of the same pathogen, accelerate epitope mapping. These tools offer a significant advantage over other methods of epitope selection because high-throughput screening can be performed in silico, followed by confirmatory studies in vitro. CTL epitopes discovered using these tools might be used to develop novel vaccines and therapeutics for the prevention and treatment of infectious diseases such as human immunodeficiency virus, hepatitis C, tuberculosis, and some cancers.     

Inhibitory effect of an antibody against alpha 1-acid glycoprotein (alpha 1-AGP) on autologous mixed lymphocyte reaction and anti-T3 T-lymphocyte activation

The action of an anti-alpha 1-AGP antibody on AMLR, anti-T3 and PHA T-lymphocyte proliferative response was evaluated. We observed a strong dose-dependent inhibition on T-lymphocyte proliferative responsiveness to autologous non-T cells and to anti-T3 stimulus, whereas PHA activation was unaffected. A lower degree of inhibition of the proliferative response was also observed on pretreating both T and non-T cells with the antibody; the addition of anti-alpha 1-AGP in the culture containing cells pretreated with the antibody showed a further inhibition of thymidine incorporation. The data suggest a direct influence of the antibody on membrane alpha 1-AGP and support a positive role of this glycoprotein (distinct from Ia and T3 antigens) on both anti-T3 and autologous non-T cell T-lymphocyte responsiveness, thus indicating the involvement of alpha 1-AGP in the T3-Ti antigen-specific pathway of T-cell activation.     

Effects of changes in membrane potential on the cyclosporin-induced inhibition of T-cell proliferation.

Cyclosporin A (CsA) exerts its major immunosuppressive effect by inhibition of T-lymphocyte proliferation. The precise mechanism and target of its action has not yet been completely identified. CsA is also known to induce a rapid membrane depolarization in T lymphocytes. We have tested the role of CsA-dependent depolarization in the inhibition of T-cell proliferation by the drug. In these studies, induced membrane depolarization (in the presence of gramicidin or by replacing the Na+ content of the medium with K+) or hyperpolarization (in the presence of valinomycin) had no influence on the induction of T-cell competence by phorbol dibutyrate/ionomycin or by submitogenic concentrations of PHA, a target for CsA immunosuppression. However, regardless of the state of membrane potential during the induction of T-cell competence, the inhibition by CsA was the same as seen in normally polarized cells. We conclude that the depolarization induced by CsA is not a critical element in its inhibitory effect on T-cell proliferation.     

T-cell responses to highly conserved CD4 and CD8 epitopes on the outer membrane protein of bovine leukemia virus: relevance to vaccine development.

Bovine leukemia virus (BLV) is a retrovirus that infects cattle and sheep and may provide a model for studying human leukemia. Cell-mediated immune mechanisms may play a major role in protection against BLV infection. We describe here for the first time the identification of proliferative (CD4) and cytotoxic T-lymphocyte (CD8) epitopes of the gp51 envelope (env) protein of BLV. This protein and a recombinant form expressed by a vaccinia virus construct have been shown to be potential vaccine candidates. A complete series of overlapping peptides, 20 amino acids in length, was prepared to identify epitopes from gp51. These peptides were tested for the ability to elicit peripheral blood lymphocyte proliferation and cytotoxic T-lymphocyte responses in infected and uninfected cattle and sheep. Peptides 51-70 and 61-80 produced a proliferative response in lymphocytes from only uninfected animals (both sheep and cattle), and this was shown by T-cell subset deletion to be a CD4-mediated response. Seven BLV-infected sheep did not show a response to either peptide. Cytotoxic T-lymphocyte activity, however, was associated only with peptides 121-140 and 131-150. In this case, the response was demonstrated to be CD8 dependent and was found only in BLV-infected animals (sheep). Knowledge of the location of these T-cell recognition domains will complement data available on B-cell epitopes in gp51 and may be useful in the design of a subunit vaccine.     

Cytotoxic-T-Lymphocyte Antigen 4 Receptor Signaling for Lymphocyte Adhesion Is Mediated by C3G and Rap1

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.     

Rearrangement and expression of T-cell antigen receptor genes in human T-lymphocyte tumor lines and normal human T-cell clones: evidence for allelic exclusion of Ti beta gene expression and preferential use of a J beta 2 gene segment.

The gene encoding the beta chain of the human T-cell receptor for antigen is composed of variable (V), diversity (D), joining (J), and constant (C) gene segments which undergo specific rearrangements during T-lymphocyte ontogeny. Southern blot analyses of seven human T-cell tumor lines and normal human T-lymphocyte clones revealed that most of these T-cell lines rearrange their Ti beta genes differently. The T-cell tumor line HPB-MLT rearranges and transcribes both of its Ti beta genes. Cloning and sequencing of the Ti beta cDNAs corresponding to these rearrangements revealed that one of the rearranged Ti beta genes is defective, while the other is functional and corresponds to the Ti beta protein expressed on the surface of these cells. Thus, this cell line displays a pattern of allelic exclusion of Ti beta gene expression. A comparison of four C beta 2-containing Ti beta cDNAs from three different cell lines revealed that three of the four utilize the same J beta 2.5 gene segment joined to different D beta and V beta genes, suggesting that there may be preferential use of this J gene during J beta 2 rearrangements. Hybridization analyses with probes for the alpha and beta genes of the T-cell receptor and the T-cell-specific T gamma gene revealed that HPB-MLT cells appear to express approximately equivalent amounts of RNA corresponding to each of the rearranged Ti alpha and Ti beta genes. However, they express a much lower level of T gamma RNA.     

The reverse stop-signal model for CTLA4 function

Activation of the T-cell co-receptor cytotoxic T-lymphocyte antigen 4 (CTLA4) has a pivotal role in adjusting the threshold for T-cell activation and in preventing autoimmunity and massive tissue infiltration by T cells. Although many mechanistic models have been postulated, no single model has yet accounted for its overall function. In this Opinion article, I outline the strengths and weaknesses of the current models, and present a new 'reverse stop-signal model' to account for CTLA4 function.     

T-cell depletion and haematological side effects of two cytotoxic monoclonal antibodies.

We investigated two cytotoxic monoclonal antibodies of BL-series (BL-IIIB4 and BL-IIG2) according to T-lymphocyte depletion from bone marrow. Both antibodies work together with human complement similar Campath-1, which was tested parallelly. The extent of T-cell depletion is about 95% for all three antibodies. On the other hand the haemopoietic side effects tested by CFU-GM recovery and LTBMC are for the BL-antibodies not as strong as for Campath-1, especially in view of LTBMC. T-cell regeneration could be shown in long term cultures. Our results indicate a possible suitability of the two investigated antibodies for T-cell depletion of bone marrow.     

T-cell differentiation antigens: proteins, genes and function

T-lymphocyte recognition, activation and function involve anumber of T-cell-specific surface proteins in addition to the receptor for antigen. The structure, function and genetic analysis of four of these T-cell differentiation antigens are discussed.     

Preferential usage of the T-cell receptor by influenza virus hemagglutinin-specific human CD4+ T lymphocytes: in vitro life span of clonotypic T cells.

Human helper T-cell (Th) responses to influenza A virus were studied by analyzing T-cell receptor V beta gene diversity in hemagglutinin-specific Th lymphocytes. The T-lymphocyte population from peripheral blood became quickly oligoclonal when stimulated in vitro with the HA306-329 peptide, and T-cell receptor V beta 3 genes were mainly expanded. Moreover, specific junctional region oligonucleotide probes corresponding to hemagglutinin-specific clones were used to estimate temporal diversity during antigenic stimulations.     

Effect of dexamethasone on T-cell receptor/CD3 expression

Glucocorticoid hormones (GCH) are anti-inflammatory and immunosuppressive agents that inhibit T-cell growth and activation. Since the T-cell receptor (TCR)/CD3 complex mediates T-lymphocyte activation, we studied the effect of in vitro dexamethasone (DEX), a synthetic GCH, on TCR/CD3 expression.DEX-treatment of a hybridoma T-cell line and normal un-transformed T-cell clones induced a decrease of the TCR/CD3 membrane expression after 4 days. After 4 weeks, TCR/CD3 was undetectable. However, the amount of mRNAs coding TCR/CD3 chains, including TCR, TCR, CD3, CD3 and CD3, as well as the amount of CD3 protein, a major component of the complex, were unaltered. By contrast, a decrease of the mRNAs deriving from the TCR gene locus, as well as of the TCR protein which is responsible for the membrane expression of the TCR/CD3 complex, was induced.These data suggest that the down-modulation of TCR expression is due to the diminution of TCR gene products in DEX-treated cells. (Mol Cell Biochem 167: 135-144, 1997)     

Paradoxical inhibition of T-cell function in response to CTLA-4 blockade; heterogeneity within the human T-cell population

T-cell co-stimulation delivered by the molecules B7-1 or B7-2 through CD28 has a positive effect on T-cell activation, whereas engagement of cytotoxic T-lymphocyte antigen 4 (CTLA-4) by these molecules inhibits activation. In vivo administration to mice of blocking monoclonal antibodies or Fab fragments against CTLA-4 can augment antigen-specific T-cell responses and, thus, therapy with monoclonal antibody against CTLA-4 has potential applications for tumor therapy and enhancement of vaccine immunization. The effects of B7-1 and B7-2 co-stimulation through CD28 depend on the strength of the signal delivered through the T-cell receptor (TCR) and the activation state of T cells during activation. Thus, we sought to determine whether these factors similarly influence the effect of B7-mediated signals delivered through CTLA-4 during T-cell activation. Using freshly isolated human T cells and Fab fragments of a monoclonal antibody against CTLA-4, we demonstrate here that CTLA-4 blockade can enhance or inhibit the clonal expansion of different T cells that respond to the same antigen, depending on both the T-cell activation state and the strength of the T-cell receptor signal delivered during T-cell stimulation. Thus, for whole T-cell populations, blocking a negative signal may paradoxically inhibit immune responses. These results provide a theoretical framework for clinical trials in which co-stimulatory signals are manipulated in an attempt to modulate the immune response in human disease.     

Systemic and Mucosal T-Lymphocyte Activation Induced by Recombinant Adenovirus Vaccines in Rhesus Monkeys

The administration of vectors designed to elicited cell-mediated immune responses may have other consequences that are clinically significant. To explore this possibility, we evaluated T-cell activation during the first 2 months after recombinant adenovirus serotype 5 (rAd5) prime or boost immunizations in rhesus monkeys. We also evaluated the kinetics of T-lymphocyte activation in both the systemic and the mucosal compartments after rAd5 administration in monkeys with preexisting immunity to Ad5. The rAd5 immunization induced lower-frequency Gag epitope-specific CD8⁺ T cells in the colonic mucosa than in the peripheral blood. There was evidence of an expansion of the simian immunodeficiency virus Gag-specific CD8⁺ T-cell responses, but not the Ad5 hexon-specific T-cell responses, following a homologous rAd5 boost. A striking but transient T-lymphocyte activation in both the systemic and the mucosal compartments of rhesus monkeys was observed after rAd5 immunization. These findings indicate that the administration of a vaccine vector such as Ad5 can induce a global activation of T cells.Considerable effort has been invested in the development of vaccine strategies for eliciting cell-mediated immune responses to human immunodeficiency virus (HIV). Studies in simian immunodeficiency virus (SIV)/SHIV-infected nonhuman primates and HIV-infected humans demonstrated a central role for cell-mediated immune responses in the containment of HIV replication (1, 12). These findings led to the hypothesis that vaccine-elicited cell-mediated immunity might contribute to improved control of HIV in infected individuals. Studies in the SIV and SHIV/macaque models have supported this hypothesis, demonstrating a decrease in peak plasma virus RNA levels during primary infection, protection against memory CD4⁺ T-cell lymphocyte loss, and prolonged survival of monkeys that had vaccine-elicited cell-mediated immunity to the virus prior to challenge (8, 15, 16).Despite promising results in preclinical nonhuman primate studies, a prophylactic HIV vaccine trial of the Merck recombinant adenovirus serotype 5 (rAd5) vector expressing HIV gag, pol, and nef genes (STEP trial) was recently halted due to a 2.3-fold increase of HIV acquisition in vaccinees with preexisting neutralizing antibodies (NAbs) to Ad5 (2, 9, 10). This finding raised the possibility that T lymphocytes that are activated in response to vaccination might represent an increased pool of potential targets for HIV infection, and the persistence of such activated cells may increase the susceptibility of the vaccinated individual to acquiring an HIV infection (5, 11). HIV replicates most readily in activated, CCR5⁺CD4⁺ T lymphocytes. It has been suggested that vaccines that elicit potent cellular immune responses may also activate subpopulations of CD4⁺ T lymphocytes. In fact, in the aftermath of the failed STEP trial, it was proposed that the activation of Ad5-specific T cells in individuals with prior Ad5 immunity may have contributed to their increased acquisition of HIV after vaccination.The contribution of cellular activation in mucosal tissues to acquisition of HIV remains unexplored (2). HIV transmission occurs most often across mucosal barriers. There is increasing evidence that CD4⁺ T lymphocytes are among the first cells infected during the transmission event (4). Activation of mucosal populations of lymphocytes as a consequence of vaccination could contribute to increasing the incidence of HIV transmission at a mucosal site.To examine these issues, the present study was initiated to explore vaccine-induced activation of T-lymphocyte populations in rhesus monkeys. The character and kinetics of the activation of both circulating and mucosal T-lymphocyte populations were evaluated after immunization with a variety of immunogens. These experiments demonstrate a striking but transient T-lymphocyte activation induced by adenovirus-based vaccine vectors in both the systemic and mucosal compartments of rhesus monkeys.     

T-lymphocyte subpopulations and tumor necrosis factor-alpha in patients with acute Flexner's dysentery

In 41 patients aged 24-63 years with acute Flexner's dysentery T-lymphocyte subpopulations CD-3, CD-4, CD-8, IRI and the content of tumor necrosis factor alpha (TNF-alpha) in the blood serum were detected. The suppression of T-cell mediated immunity on account of the presence of CD-4 with decreased immunoregulatory index and an increased content of TNF-alpha were established. Changes in T-cell mediated immunity and TNF-alpha depended on the period and the severity of the diseases.